S154: IDENTIFICATION OF NOVEL FACTORS CONTROLLING NON GENETIC CELL PLASTICITY IN CHRONIC MYELOID LEUKEMIA
Authors: Guido Baselli, Alisa Alekseenko, Eva Brinkman, Yerma Pareja-Sanchez, Lefteris Sinanis, Vicent Pelechano
Journal: HemaSphere (2023)

Background: In cancer cell communities the dynamic fluctuation of gene expression can lead some cells to acquire rare drug resistant phenotypes. These cells can survive even targeted therapies impairing our ability to eradicate the disease. This phenotypic plasticity has been observed in several tumors and is now recognized as a hallmark of cancer. In the K562 chronic myeloid leukaemia (CML) cells it has been identified a drug resistant subpopulation overexpressing the surface marker CD24. Importantly, the CD24+ phenotype was shown to be reversible as both CD24+ and - cells were able to originate a heterogeneous culture over time. Aims: Aim of this study will be to leverage the dynamic expression of the CD24 in K562 CML cells to track these phenotypic state transitions and to identify genes regulating this dynamic by genome wide CRISPR screening. Then we aim to investigate these genes association with drug resistance in CML. Methods: Genome wide CRISPR-KO screening was performed K562 cells using the Brunello genome-wide sgRNA library. To isolate cells changing their phenotype we integrated the results of two different approaches. In the first experiment (Figure, upper panel) we sorted CD24+ and - cells sequentially by magnetic activated cell sorting to retrospectively identify the cells changing their phenotype. In the second experiment (Figure, lower panel) we leveraged the temporal delay between mRNA and protein levels variation upon state transition. Briefly, we measured mRNA and protein levels by combining mRNA fluorescence in situ hybridization (FISH) and protein staining. Cells showing inconsistent levels of mRNA and protein were identified as state transitioning cells. A custom analysis pipeline was designed to identify genes associated with state transition in the two screenings by multiplicity adjusted negative binomial regression. Differential gene expression analysis in CML patients was performed by multiplicity adjusted Wilcoxon test. Analyses were performed using R v4.0.3, adjusted p<0.1 were considered statistically significant. Results: By CRIPSR screening, we were able to associate 49 genes with phenotypic plasticity in K562 cells. Of those 32 were depleted in KO events in the switching cells and therefore we hypothesize these genes to promote the phenotypic transition. Conversely, the other 17 were enriched in KO events in the switching cells and therefore associated to a regulatory role. We then assessed the impact of the 49 putative plasticity regulators on drug sensitivity in CML patients. To this extent we leverage publicly available data from 96 CML patients undergoing PBMC transcriptome profiling at the diagnosis and then starting a therapy with Imatinib. Early molecular response (EMR) failure at month 3 of therapy was used as a proxy for Imatinib resistance. Genes associated with a faster transition dynamic in the CRISPR screening were expected to be overexpressed while the putative negative regulator of plasticity was expected to be repressed. We associated 7 genes with drug resistance in these patients. Interestingly, those genes were mainly involved in the regulation of cell energetic homeostasis. Indeed, among the negative regulator of plasticity, we found a key enzyme of glycolysis and a lysosomal peptidase and among the negative regulators of this process we found three genes involved in oxidative metabolism. Summary/Conclusion: Overall, we identified a gene signature associated with the regulation of cell plasticity in CML models and drug resistance in the patients. Interestingly, our results suggest a link between cell plasticity and the regulation of cell energetic metabolism in CML.Keywords: Chronic myeloid leukemia, Drug resistance, Plasticity